The diatom Phaeodactylum tricornutum adjusts nonphotochemical fluorescence quenching capacity in response to dynamic light via fine-tuned Lhcx and xanthophyll cycle pigment synthesis.

Diatoms contain a highly flexible capacity to dissipate excessively absorbed light by nonphotochemical fluorescence quenching (NPQ) based on the light-induced conversion of diadinoxanthin (Dd) into diatoxanthin (Dt) and the presence of Lhcx proteins. Their NPQ fine regulation on the molecular level upon a shift to dynamic light conditions is unknown. We investigated the regulation of Dd + Dt amount, Lhcx gene and protein synthesis and NPQ capacity in the diatom Phaeodactylum tricornutum after a change from continuous low light to 3 d of sine (SL) or fluctuating (FL) light conditions. Four P. tricornutum strains with different NPQ capacities due to different expression of Lhcx1 were included. All strains responded to dynamic light comparably, independently of initial NPQ capacity. During SL, NPQ capacity was strongly enhanced due to a gradual increase of Lhcx2 and Dd + Dt amount. During FL, cells enhanced their NPQ capacity on the first day due to increased Dd + Dt, Lhcx2 and Lhcx3; already by the second day light acclimation was accomplished. While quenching efficiency of Dt was strongly lowered during SL conditions, it remained high throughout the whole FL exposure. Our results highlight a more balanced and cost-effective photoacclimation strategy of P. tricornutum under FL than under SL conditions.

High light acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool.

In diatoms, the process of energy-dependent chlorophyll fluorescence quenching (qE) has an important role in photoprotection. Three components are essential for qE: (1) the light-dependent generation of a transthylakoidal proton gradient; (2) the deepoxidation of the xanthophyll diadinoxanthin (Dd) into diatoxanthin (Dt); and (3) specific nucleus-encoded antenna proteins, called Light Harvesting Complex Protein X (LHCX). We used the model diatom Phaeodactylum tricornutum to investigate the concerted light acclimation response of the qE key components LHCX, proton gradient, and xanthophyll cycle pigments (Dd+Dt) and to identify the intracellular light-responsive trigger. At high-light exposure, the up-regulation of three of the LHCX genes and the de novo synthesis of Dd+Dt led to a pronounced rise of qE. By inhibiting either the conversion of Dd to Dt or the translation of LHCX genes, qE amplification was abolished and the diatom cells suffered from stronger photoinhibition. Artificial modification of the redox state of the plastoquinone (PQ) pool via 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone resulted in a disturbance of Dd+Dt synthesis in an opposite way. Moreover, we could increase the transcription of two of the four LHCX genes under low-light conditions by reducing the PQ pool using 5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone. Altogether, our results underline the central role of the redox state of the PQ pool in the light acclimation of diatoms. Additionally, they emphasize strong evidence for the existence of a plastid-to-nucleus retrograde signaling mechanism in an organism with plastids that derived from secondary endosymbiosis.

A novel type of light-harvesting antenna protein of red algal origin in algae with secondary plastids.

BACKGROUND: Light, the driving force of photosynthesis, can be harmful when present in excess; therefore, any light harvesting system requires photoprotection. Members of the extended light-harvesting complex (LHC) protein superfamily are involved in light harvesting as well as in photoprotection and are found in the red and green plant lineages, with a complex distribution pattern of subfamilies in the different algal lineages. RESULTS: Here, we demonstrate that the recently discovered "red lineage chlorophyll a/b-binding-like proteins" (RedCAPs) form a monophyletic family within this protein superfamily. The occurrence of RedCAPs was found to be restricted to the red algal lineage, including red algae (with primary plastids) as well as cryptophytes, haptophytes and heterokontophytes (with secondary plastids of red algal origin). Expression of a full-length RedCAP:GFP fusion construct in the diatom Phaeodactylum tricornutum confirmed the predicted plastid localisation of RedCAPs. Furthermore, we observed that similarly to the fucoxanthin chlorophyll a/c-binding light-harvesting antenna proteins also RedCAP transcripts in diatoms were regulated in a diurnal way at standard light conditions and strongly repressed at high light intensities. CONCLUSIONS: The absence of RedCAPs from the green lineage implies that RedCAPs evolved in the red lineage after separation from the the green lineage. During the evolution of secondary plastids, RedCAP genes therefore must have been transferred from the nucleus of the endocytobiotic alga to the nucleus of the host cell, a process that involved complementation with pre-sequences allowing import of the gene product into the secondary plastid bound by four membranes. Based on light-dependent transcription and on localisation data, we propose that RedCAPs might participate in the light (intensity and quality)-dependent structural or functional reorganisation of the light-harvesting antennae of the photosystems upon dark to light shifts as regularly experienced by diatoms in nature. Remarkably, in plastids of the red lineage as well as in green lineage plastids, the phycobilisome based cyanobacterial light harvesting system has been replaced by light harvesting systems that are based on members of the extended LHC protein superfamily, either for one of the photosystems (PS I of red algae) or for both (diatoms). In their proposed function, the RedCAP protein family may thus have played a role in the evolutionary structural remodelling of light-harvesting antennae in the red lineage.

Silencing of the violaxanthin de-epoxidase gene in the diatom Phaeodactylum tricornutum reduces diatoxanthin synthesis and non-photochemical quenching.

Diatoms are a major group of primary producers ubiquitous in all aquatic ecosystems. To protect themselves from photooxidative damage in a fluctuating light climate potentially punctuated with regular excess light exposures, diatoms have developed several photoprotective mechanisms. The xanthophyll cycle (XC) dependent non-photochemical chlorophyll fluorescence quenching (NPQ) is one of the most important photoprotective processes that rapidly regulate photosynthesis in diatoms. NPQ depends on the conversion of diadinoxanthin (DD) into diatoxanthin (DT) by the violaxanthin de-epoxidase (VDE), also called DD de-epoxidase (DDE). To study the role of DDE in controlling NPQ, we generated transformants of P. tricornutum in which the gene (Vde/Dde) encoding for DDE was silenced. RNA interference was induced by genetic transformation of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or, alternatively, with a plasmid mediating the expression of a self-complementary hairpin-like construct (inverted repeat, IR). The silencing approaches generated diatom transformants with a phenotype clearly distinguishable from wildtype (WT) cells, i.e. a lower degree as well as slower kinetics of both DD de-epoxidation and NPQ induction. Real-time PCR based quantification of Dde transcripts revealed differences in transcript levels between AS transformants and WT cells but also between AS and IR transformants, suggesting the possible presence of two different gene silencing mediating mechanisms. This was confirmed by the differential effect of the light intensity on the respective silencing efficiency of both types of transformants. The characterization of the transformants strengthened some of the specific features of the XC and NPQ and confirmed the most recent mechanistic model of the DT/NPQ relationship in diatoms.

FIRST INDUCED PLASTID GENOME MUTATIONS IN AN ALGA WITH SECONDARY PLASTIDS: psbA MUTATIONS IN THE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) REVEAL CONSEQUENCES ON THE REGULATION OF PHOTOSYNTHESIS(1).

Diatoms play a crucial role in the biochemistry and ecology of most aquatic ecosystems, especially because of their high photosynthetic productivity. They often have to cope with a fluctuating light climate and a punctuated exposure to excess light, which can be harmful for photosynthesis. To gain insight into the regulation of photosynthesis in diatoms, we generated and studied mutants of the diatom Phaeodactylum tricornutum Bohlin carrying functionally altered versions of the plastidic psbA gene encoding the D1 protein of the PSII reaction center (PSII RC). All analyzed mutants feature an amino acid substitution in the vicinity of the QB -binding pocket of D1. We characterized the photosynthetic capacity of the mutants in comparison to wildtype cells, focusing on the way they regulate their photochemistry as a function of light intensity. The results show that the mutations resulted in constitutive changes of PSII electron transport rates. The extent of the impairment varies between mutants depending on the proximity of the mutation to the QB -binding pocket and/or to the nonheme iron within the PSII RC. The effects of the mutations described here for P. tricornutum are similar to effects in cyanobacteria and green microalgae, emphasizing the conservation of the D1 protein structure among photosynthetic organisms of different evolutionary origins.